Method and system for optical stimulation of neurons

ABSTRACT

A method of stimulating neurons present in a living body is disclosed. The method comprises directing light to an artificial light absorbing medium implanted extracellularly at a target location having the neurons therein, wherein wavelengths and intensities of the light are selected so as to heat the light absorbing medium by light absorption. The heating is sufficient to stimulate neurons nearby the light absorbing medium. In some embodiments, the light is spatially modulating so as to encode a stimulation pattern therein.

RELATED APPLICATIONS

This application is a division of U.S. patent application Ser. No.12/746,525 filed on Jun. 6, 2010, which is a National Phase of PCTPatent Application No. PCT/IL2008/001574 having International filingdate of Dec. 4, 2008, which claims the benefit of priority of U.S.Provisional Patent Application Nos. 61/089,713 filed on Aug. 18, 2008and 60/992,921 filed on Dec. 6, 2007. The contents of the aboveapplications are all incorporated herein by reference.

FIELD AND BACKGROUND OF THE INVENTION

The present invention, in some embodiments thereof, relates to neuronstimulation and, more particularly, but not exclusively, to lightinduced neuron stimulation.

A neuroprosthesis is a device which is designed for replacing orimproving the function of an impaired nervous system or sensory organ.Known in the art are neuroprostheses for the treatment of functionaldisorders of the visual system, hearing system, cranial nerve system,spinal cord and peripheral nervous system [to this end see, e.g.,International Patent Publication Nos. 98/036793 and WO 98/036795, andU.S. Pat. Nos. 6,393,327, 6,497,699, 6,829,510 and 7,447,548].

A visual prosthesis, for example, is a device that captures aspects ofthe visual environment and uses this information to stimulate nerveswithin the visual pathway to influence vision. A visual prosthesis maybe placed within the eye or at some location on the path toward orwithin the visual part of the brain. Visual prosthetic devices withinthe eye can be positioned on the inner surface of the retina (i.e.,epi-retinal) or under the retina (sub-retinal).

An auditory prosthesis is a device that delivers stimuli representativeof sound to the spiral ganglion which is responsible for transmittingimpulses from the inner ear to the brain. Also known are auditoryprostheses that deliver stimuli directly to the auditory cortex or theauditory brainstem.

A neuroprosthesis operates by interacting with neurons. A neuronconsists of: a branched pattern of processes, commonly known asdendrites, which act to receive information; a cell body, known as thesoma, from which the dendrites extend, and which integrates the receivedinformation and provides for the metabolic needs of the neuron; and anaxon extending from the soma, for transporting constituents between thesoma and distant synapses, which transfer information to the next set ofnerve dendrites.

When no stimulation is presented, the neuron is negatively polarizedinside the soma membrane with respect to the outside of the membrane.Depolarization of a soma membrane creates an action potential, whicheffectively travels via axons, e.g., to the inner brain, thereby sendingthe stimulation signal thereto. Thus, information is represented in thenervous system as a series of action potentials that travel between theneurons via the membranes of axons.

Once stimulated, the neuron generates and allows propagation of anelectrical impulse therein. Various techniques have been utilized forstimulating neurons. These include electrical stimulation, mechanicalstimulation, thermal stimulation, chemical stimulation and opticalstimulation. In the field of neuroprostheses, the most commonstimulation technique is electrical stimulation wherein a transientcurrent or voltage pulse is applied via electrodes. For example, manyvisual prostheses include epi-retinal or sub-retinal microelectrodearray implants, manufactured using micro-fabrication technology borrowedfrom the semiconductor industry.

Published Application No. 20030208245 discloses a technique for directlystimulating neural tissue with optical energy. An optical field having awavelength of 1-6 μm is focused on a target neural tissue such that thetarget neural tissue propagates an electrical impulse. The focusing isonto an area of 50-600 μm micrometers. U.S. Published Application No.20060161227 discloses a system for stimulating auditory neuronsassociated with the spiral ganglion cells. Optical energy at awavelength of 0.5-10 μm is delivered along an optical path to a targetsite of auditory neurons to evoke compound action potential therein. Theevoked action potential is monitored by monitoring means.

The light sources used in the above publications are laser devices, andthe produced optical energy is absorbed by the tissue's water contentwithin a typical absorption distance of several hundred microns,allowing the optical field to directly interact with the tissue. It hasbeen hypothesized [Wells et al., “Biophysical mechanisms of transientoptical stimulation of peripheral nerve,” Biophys J 93, 2567-80 (2007)]that the photobiological effect of light absorption on the tissue ismediated through a photo-thermal mechanism, rather than electricalfield, photochemical or photomechanical mechanisms.

A system capable of patterned activation of many neurons withmillisecond precision using rapid UV laser deflection has recently beendisclosed [Shoham et al., “Rapid neurotransmitter uncaging in spatiallydefined patterns,” Nature Methods 2, 837-843 (2005)]. In this system, UVlight is used to activate neurons by uncaging glutamate, the majorexcitatory neurotransmitter in the central nervous system.

Recently, retinal ganglion cells have been directly activated byartificially causing them to express Channelrhodopsin II (ChR2), alight-gated cation channel [Reutsky et al., “Patterned opticalactivation of Channelrhodopsin II expressing retinal ganglion cells,” inCNE '07. 3rd International IEEE/EMBS Conference on Neural Engineering,2007 50-52 (2007)]. Patterned stimulation of the cells was demonstratedby means of video projection technology based on a Texas InstrumentsDigital Mirror Device.

Additional background art includes Wells et al. “Optical stimulation ofneural tissue in vivo,” Opt Lett 30, 504-6 (2005); Pappas et al.,“Nanoscale Engineering of a Cellular Interface with SemiconductorNanoparticle Films for Photoelectric Stimulation of Neurons,” NanoLetters (2007) Vol. 7, No. 2, 513-519 and Izzo et al., “Laserstimulation of the auditory nerve,” Lasers Surg Med 38, 745-53 (2006);U.S. Published Application Nos. 20060161227 and 20030208245].

SUMMARY OF THE INVENTION

According to an aspect of some embodiments of the present inventionthere is provided a method of stimulating neurons present in a livingbody. The method comprises directing light to an artificial lightabsorbing medium implanted extracellularly at a target location havingthe neurons therein, wherein wavelengths and intensities of the lightare selected so as to heat the light absorbing medium by lightabsorption, the heating being sufficient to stimulate neurons nearby thelight absorbing medium.

According to some embodiments of the invention the method furthercomprises extracellularly implanting the artificial light absorbingmedium at the target location.

According to some embodiments of the invention the light is directed soas to simultaneously form a stimulation pattern at the target location.

According to some embodiments of the invention the method furthercomprises spatially modulating the light so as to encode the stimulationpattern therein.

According to an aspect of some embodiments of the present inventionthere is provided a method of stimulating neurons. The method comprises:spatially modulating light so as to encode a stimulation patterntherein; and directing the light to a target location having the neuronstherein so as to simultaneously form the stimulation pattern at thetarget location, wherein wavelengths and intensities forming thestimulation pattern are selected so as to selectively stimulate theneurons.

According to an aspect of some embodiments of the present inventionthere is provided a neurostimulation system. The neurostimulation systemcomprises: an artificial light absorbing medium implantableextracellularly in a living body at target location having therein aplurality of neurons; and an illumination system having a light sourcefor generating light and optics for directing the light to theartificial light absorbing medium, wherein wavelengths and intensitiesof the light are selected so as to heat the light absorbing medium bylight absorption, the heating being sufficient to stimulate neuronsnearby the light absorbing medium.

According to some embodiments of the invention the illumination systemcomprises a projector system configured for generating a spatiallymodulated light beam encoded with a stimulation pattern.

According to an aspect of some embodiments of the present inventionthere is provided a neurostimulation device. The neurostimulation devicecomprises: a projector system for generating a spatially modulated lightbeam encoded with a stimulation pattern; and optics configured fordirecting the light to a target location having a plurality of neuronsso as to simultaneously form the stimulation pattern at the targetlocation; wherein wavelengths and intensities forming the stimulationpattern are selected so as to selectively stimulate the neurons.

According to an aspect of some embodiments of the present inventionthere is provided a neuroprosthesis system. The neuroprosthesis systemcomprises: the neurostimulation device described herein; and a sensingdevice for sensing information from the environment and transmittingsignals pertaining to the information to the neurostimulation device;wherein the projector system is configured for calculating thestimulation pattern based on the information.

According to some embodiments of the invention the target location isimplanted with an artificial light absorbing medium, and wherein thewavelengths and intensities are selected so as to heat the lightabsorbing medium by light absorption, the heating being sufficient tostimulate neurons nearby the light absorbing medium.

According to some embodiments of the invention the neurons express alight-activated ion channel protein, and wherein the wavelengths andintensities are selected so as to activate the light-activated ionchannel protein.

According to some embodiments of the invention the method furthercomprises, transfecting the neurons by gene transfer vectors capable ofinducing expression of the light-activated ion channel protein.

According to some embodiments of the invention the projector systemcomprises a spatial light modulator having a liquid crystal.

According to some embodiments of the invention the projector system isconfigured for providing phase-only modulation.

According to some embodiments of the invention the projector system isconfigured for providing concurrent phase and amplitude modulation.

According to some embodiments of the invention the optics is afree-space optics.

According to some embodiments of the invention the optics comprises anoptical fiber bundle.

According to some embodiments of the invention the light absorbingmedium is extracellularly distributed at the target location.

According to some embodiments of the invention the light absorbingmedium comprises light absorbing particles.

According to some embodiments of the invention the light absorbingmedium comprises a dye.

According to some embodiments of the invention the light absorbingparticles are metallic nanoparticles.

According to some embodiments of the invention the light absorbingmedium comprises a light absorbing film.

According to some embodiments of the invention the stimulation patternis a three-dimensional stimulation pattern.

According to some embodiments of the invention the light is selected soas to generate non-linear optical effects.

According to some embodiments of the invention the light ischaracterized by a pulse width which is shorter that one picosecond.

According to some embodiments of the invention the target location is aretina.

According to some embodiments of the invention the target location is acochlea.

According to some embodiments of the invention the target location is inthe cerebral cortex.

According to some embodiments of the invention the target location is inthe brainstem.

According to some embodiments of the invention the target location isthe vagus nerve.

According to some embodiments of the invention the target location is acranial nerve.

According to some embodiments of the invention the target location is aneuron culture.

According to some embodiments of the invention the modulated light beamis dynamically encoded with an alternating sequence of pseudo-randomframes forming together a symmetric stimulation pattern at the targetlocation.

According to some embodiments of the invention the intensities areselected so as to reduce efficiency inhomogeneities within thestimulation pattern.

According to some embodiments of the invention the intensities areinversely proportional to a square of a sinc function.

According to some embodiments of the invention the optics ischaracterized by a numerical aperture selected such that an elementaryspot size of the stimulation pattern is at most 20 microns.

According to some embodiments of the invention the light is encoded in atime-multiplexed manner, wherein stimulation patterns transmitted atdifferent times correspond to different light spectra.

Unless otherwise defined, all technical and/or scientific terms usedherein have the same meaning as commonly understood by one of ordinaryskill in the art to which the invention pertains. Although methods andmaterials similar or equivalent to those described herein can be used inthe practice or testing of embodiments of the invention, exemplarymethods and/or materials are described below. In case of conflict, thepatent specification, including definitions, will control. In addition,the materials, methods, and examples are illustrative only and are notintended to be necessarily limiting.

Implementation of the method and/or system of embodiments of theinvention can involve performing or completing selected tasks manually,automatically, or a combination thereof. Moreover, according to actualinstrumentation and equipment of embodiments of the method and/or systemof the invention, several selected tasks could be implemented byhardware, by software or by firmware or by a combination thereof usingan operating system.

For example, hardware for performing selected tasks according toembodiments of the invention could be implemented as a chip or acircuit. As software, selected tasks according to embodiments of theinvention could be implemented as a plurality of software instructionsbeing executed by a computer using any suitable operating system. In anexemplary embodiment of the invention, one or more tasks according toexemplary embodiments of method and/or system as described herein areperformed by a data processor, such as a computing platform forexecuting a plurality of instructions. Optionally, the data processorincludes a volatile memory for storing instructions and/or data and/or anon-volatile storage, for example, a magnetic hard-disk and/or removablemedia, for storing instructions and/or data. Optionally, a networkconnection is provided as well. A display and/or a user input devicesuch as a keyboard or mouse are optionally provided as well.

BRIEF DESCRIPTION OF THE DRAWINGS

Some embodiments of the invention are herein described, by way ofexample only, with reference to the accompanying drawings. With specificreference now to the drawings in detail, it is stressed that theparticulars shown are by way of example and for purposes of illustrativediscussion of embodiments of the invention. In this regard, thedescription taken with the drawings makes apparent to those skilled inthe art how embodiments of the invention may be practiced.

In the drawings:

FIG. 1 is a flowchart diagram of a method suitable for stimulatingneurons present in a living body, according to various exemplaryembodiments of the present invention;

FIG. 2 is a flowchart diagram of another method suitable for stimulatingneurons present in a living body, according to various exemplaryembodiments of the present invention;

FIG. 3 is a flowchart diagram of an additional method suitable forstimulating neurons present in a living body, according to variousexemplary embodiments of the present invention;

FIG. 4 is a flowchart diagram of a further method suitable forstimulating neurons present in a living body, according to variousexemplary embodiments of the present invention;

FIGS. 5A-E are schematic illustrations of a neurostimulation system,according to various exemplary embodiments of the present invention;

FIG. 6 is a schematic illustration a neurostimulation device, accordingto various exemplary embodiments of the present invention;

FIG. 7 is a schematic illustration of a neuroprosthesis system,according to various exemplary embodiments of the present invention;

FIGS. 8A-C are schematic illustrations of an experimental setup (FIG.8A) and procedure (FIGS. 8B-C), utilized in experiments performed inaccordance with some embodiments of the present invention;

FIG. 8D shows a phase image used in experiments, performed in accordancewith some embodiments of the present invention, for generating a sparsepseudo-random pattern of photo-stimulation points;

FIGS. 9A-D shows theoretical and measured spatial distribution ofefficiency, as predicted and measured in experiments performed inaccordance with some embodiments of the present invention;

FIG. 9E shows intensities of individual stimulation shapes as controlledaccording to various exemplary embodiments of the present invention;

FIG. 9F shows average power density per stimulation shape, according tovarious exemplary embodiments of the present invention;

FIG. 10A is a three-dimensional plot of Gaussian stimulation shapeaccording to various exemplary embodiments of the present invention;

FIGS. 10B-C demonstrate techniques for controlling the size of thestimulation shape;

FIG. 10D demonstrates techniques for reducing speckle, according tovarious exemplary embodiments of the present invention;

FIG. 10E show a characteristic stimulation pulse, according to variousexemplary embodiments of the present invention;

FIG. 10F shows time sharing technique utilizing the shortness of thepulse of FIG. 10E, according to various exemplary embodiments of thepresent invention;

FIG. 11A shows raw data of a single neuron response, as measured inexperiments performed in accordance with some embodiments of the presentinvention;

FIG. 11B shows responses of three different neurons as measured inexperiments performed in accordance with some embodiments of the presentinvention; and

FIGS. 12A-D show a spatial (FIGS. 12A-C) and temporal (FIG. 12D) thermalresponse profile, as obtained by computer simulations, performed inaccordance with some embodiments of the present invention; and

FIG. 13 is an image of a stained retina, according to some embodimentsof the present invention.

DESCRIPTION OF SPECIFIC EMBODIMENTS OF THE INVENTION

The present invention, in some embodiments thereof, relates to neuronstimulation and, more particularly, but not exclusively, to lightinduced neuron stimulation.

Before explaining at least one embodiment of the invention in detail, itis to be understood that the invention is not necessarily limited in itsapplication to the details of construction and the arrangement of thecomponents and/or methods set forth in the following description and/orillustrated in the drawings and/or the Examples. The invention iscapable of other embodiments or of being practiced or carried out invarious ways.

Some embodiments of the present invention are directed to a method,device and system suitable for stimulating neurons present in a body ofliving subject, typically a mammalian subject, e.g., a human subject. Invarious exemplary embodiments of the invention the neurons arestimulated by optical energy delivered to a target location at which theneurons are present.

The target location can be any location in the living body in whichthere is a neural tissue containing a large population of neurons. Insome embodiments of the present invention the target location is aretina of the subject, in which case the neurons to be stimulated canbe, for example, the retinal ganglion cells. In some embodiments of thepresent invention the target location is part of the cochlea of thesubject, in which case the neurons to be stimulated are the spiralganglion cells. In some embodiments of the present invention the targetlocation is part of the cerebral cortex of the subject's brain,preferably the visual cortex or the auditory cortex, but may also be orinclude other cortical regions such as, but not limited to, motorcortex, premotor cortex, supplementary motor cortex, somatosensorycortex, etc. In some embodiments of the present invention the targetlocation is one or more of the brainstem structures (midbrain, pons,medulla) of the subject. In some embodiments of the present inventionthe target location is part of the Thalamus or the sub-Thalamic nucleus(STN). In some embodiments of the present invention the target locationis a nerve in the peripheral nervous system, the autonomic nervoussystem or cranial nerve system, e.g.: the vagus nerve, the optic nerve,the auditory nerve, a motor nerve, or a sensory nerve. In someembodiments of the present invention the target location is part of thespinal cord of the subject. In some embodiments of the present inventionthe target location is part of the enteric nervous system.

The target location can also be an ex-vivo target location, such as, butnot limited to, a neuron culture.

Referring now to the drawings, each of FIGS. 1-4 is a flowchart diagramof a method suitable for stimulating neurons present in a living body,according to various exemplary embodiments of the present invention.

It is to be understood that, unless otherwise defined, the operationsdescribed hereinbelow can be executed either contemporaneously orsequentially in many combinations or orders of execution. Specifically,the ordering of any of the flowchart diagrams in the drawings is not tobe considered as limiting. For example, two or more operations,appearing in the following description or in the flowchart diagrams in aparticular order, can be executed in a different order (e.g., a reverseorder) or substantially contemporaneously. Additionally, severaloperations described below are optional and may not be executed.

With specific reference to FIG. 1, the method, referred to hereinunderas method 10, begins at 11 and optionally and preferably continues to 12at which a light absorbing medium is implanted in a target locationhaving the neurons therein. Alternatively, the method can be executedafter the light absorbing medium has been implanted in the targetlocation.

The light absorbing medium can be in the form of a plurality ofparticles, such as biocompatible dyes or physiologically acceptablenanoparticles having sufficiently high light absorption coefficients insitu (preferably above 10 mm⁻¹, more preferably above 30 mm⁻¹, morepreferably above 40 mm⁻¹, more preferably above 50 mm⁻¹ across awavelength range spanning from IR to Violet or at specific absorptionpeaks within this range). Representative examples of such particlesinclude, without limitation, various biocompatible inks (e.g., Indiaink, or any of the inks disclosed in U.S. Pat. No. 7,288,578 and U.S.Published Application No. 20080012164) and light-absorbingnanoparticles, for example, metallic (e.g., gold) nanoparticles.

The light absorbing medium can comprise a pigment or a dye. One group ofpigments suitable for the present embodiments is the family of pigmentsused for Tattooing, which are known to be biocompatible. There are anumber of Tattoo ink types which come in various colors. Black dyesinclude India ink, which is made from carbon particles. India ink haspreviously been used as an optical absorber in tissue-simulatingphantoms. Other black inks are commonly made from powdered minerals andcrystals, and amorphous carbon from combustion. Also contemplated areother colors, such as, but not limited to, brown inks, e.g., inks madefrom ion oxides and clay, red inks, e.g., inks are made from Napthaderivatives, orange and/or yellow inks, e.g., inks made from Monoazo andDiazo synthetic pigments, purples inks, e.g., inks made from Dioxazine,blue and/or green inks, e.g., inks made from copper Pthyocalanine.

The light absorbing medium can comprise nanoparticles, such as, but notlimited to, gold and/or silver and/or titanium oxide nanoparticles whichare highly absorbing in the visible range. The peak absorption around530 nm of 10 nm gold particles is red-shifted when the size of theparticles becomes larger. The light absorbing medium can also comprisenanorods, e.g., gold nanorods which are rod-shaped gold nanoparticleswhose aspect ratios tune the surface plasmon resonance (SPR) band fromthe visible to near infrared wavelength. The use of nanorods isadvantageous because near infrared light transmits readily through humanskin and tissue.

The light absorbing medium can also comprise a biological material. Onetype of biological light absorbing medium is melanin, which is aheterogeneous, complex polymer that is synthesized in specializedorganelles (melanosomes) of the epidermal melanocytes and thentransferred to surrounding keratinocytes. The melanin absorbs lightacross the entire visible range and into the near-IR. Another family ofbiological light absorbing medium are porphyrins, which are composed ofa deeply colored heterocyclic macrocycle characterized by the presenceof four pyrroline subunits interconnected via their a carbon atoms viamethine bridges (for example: heme and chlorophyll). Another type ofbiological light absorbing medium is retinoids, which is a chemicalcompound that is chemically related to vitamin A. Representativeexamples of retinoids suitable for the present embodiment, include,without limitation, retinal and beta-carotene. Another type ofbiological light absorbing medium is bacteriorhodopisin, which absorbslight at a wavelength range of 500-650 nm (absorption maximum at 568nm).

Other types of light absorbing media are colorants that are presentlywidely used in various food, drugs and cosmetics (FD&C) applications.The colorants can optionally be encapsulated in a sol jel matrix forprolonging their presence in the target location. Such encapsulationtechnique is known in the art, see, e.g., International PatentApplication Nos. WO01/80823 and WO04/081222. A review of sol jeltechnique is found in Avnir et al., in The Chemistry of OrganosiliconCompounds, Vol. 2 (1998), Chapter 48 Page 192.

Representative examples of water-soluble colorants that are usable inthis and other aspects of the present invention include, withoutlimitation, FD&C colors such as EXT. D&C Green No. 1, EXT. D&C YellowNo. 7, EXT. D&C Yellow No. 1, EXT. D&C Orange No. 3, FD&C Red No. 4, D&COrange No. 4, FD&C Yellow No. 6, D&C Red No. 2, D&C Red No. 33, EXT. D&CYellow No. 3, FD&C Yellow No. 5, D&C Brown No. 1, D&C Black No. 1, FD&CGreen No. 3, FD&C Blue No. 1, D&C Blue No. 4, D&C Red No. 19, D&C RedNo. 37, EXT. D&C Red No. 3, D&C Yellow No. 8, D&C Orange No. 5, D&C RedNo. 21, D&C Red No. 22, D&C Red No. 28, D&C Red No. 27, D&C Orange No.10, D&C Orange No. 11, FD&C Red No. 3, D&C Yellow No. 11, D&C Yellow No.10, D&C Green No. 8, EXT. D&C Violet No. 2, D&C Green No. 5 and FD&CBlue No. 2.

A list of additional water-soluble colorants usable in the context ofthe present embodiments, according to their Color Index No. and name,their Japanese name and their FDA name is set forth in Table 1 below.

TABLE 1 CI No. Japanese name FDA name Color Index name C.I.10020 GreenNo. 401 (EXT.D & C Green No. 1) Acid Green 1 C.I.10316 Yellow No. 403-1(EXT.D & C Yellow No. 7) Acid Yellow 1 C.I.13065 Yellow No. 406 (EXT.D &C Yellow No. 1) Acid Yellow 36 C.I.14600 Orange No. 402 (EXT. D & COrange No. 3) Acid Orange 20 C.I.14700 Red No. 504 FD & C Red No. 4 FoodRed 1 C.I.15510 Orange No. 205 D & C Orange No. 4 Acid Orange 7C.I.15620 Red No. 506 Acid Red 88 C.I.15985 Yellow No. 5 FD & C YellowNo. 6 Food Yellow 3 C.I.16150 Red No. 503 Acid Red 26 C.I.16155 Red No.502 Food Red 6 C.I.16185 Red No. 2 (D & C Red No. 2) Acid Red 27C.I.16255 Red No. 102 Acid Red 18 C.I.17200 Red No. 227 D & C Red No. 33Acid Red 33 C.I.18820 Yellow No. 407 (EXT.D & C Yellow No. 3) AcidYellow 11 C.I.18950 Yellow No. 402 Acid Yellow 40 C.I.19140 Yellow No. 4FD & C Yellow No. 5 Acid Yellow 23 C.I.20170 Brown No. 201 (D & C BrownNo. 1) Acid Orange 24 C.I.20470 Black No. 401 (D & C Black No. 1) AcidBlack 1 C.I.42052 Blue No. 202 Acid Blue 5 C.I.42052 Blue No. 203 AcidBlue 5 C.I.42053 Green No. 3 FD & C Green No. 3 Food Green C.I.42085Green No. 402 Acid Green 3 C.I.42090 Blue No. 1 FD & C Blue No. 1 FoodBlue 2 C.I.42090 Blue No. 205 D & C Blue No. 4 Acid Blue 9 C.I.42095Green No. 205 Acid Green 5 C.I.45100 Red No. 106 Acid Red 52 C.I.45170Red No. 213 (D & C Red No. 19) Basic Violet 10 C.I.45170 Red No. 214Solv. Red 49 C.I.45170 Red No. 215 (D & C Red No. 37) Solv. Red 49C.I.45190 Red No. 401 (EXT. D & C Red No. 3) Acid Violet 9 C.I.45350Yellow No. 202-1 D & C Yellow No. 8 Acid Yellow 73 C.I.45350 Yellow No.202-2 Acid Yellow 73 C.I.45370 Orange No. 201 D & C Orange No. 5 Solv.Red 72 C.I.45380 Red No. 223 D & C Red No. 21 Solv. Red 43 C.I.45380 RedNo. 230-1 D & C Red No. 22 Acid Red 87 C.I.45380 Red No. 230-2 Acid Red87 C.I.45410 Red No. 104-1 D & C Red No. 28 Acid Red 92 C.I.45410 RedNo. 218 D & C Red No. 27 Solv. Red 48 C.I.45410 Red No. 231 Acid Red 92C.I.45425 Orange No. 206 D & C Orange No. 10 Solv. Red 73 C.I.45425Orange No. 207 D & C Orange No. 11 Acid Red 95 C.I.45430 Red No. 3 FD &C Red No. 3 Acid Red 51 C.I.45440 Red No. 105-1 Acid Red 94 C.I.45440Red No. 232 Acid Red 94 C.I.47000 Yellow No. 204 D & C Yellow No. 11Solv. Yellow 33 C.I.47005 Yellow No. 203 D & C Yellow No. 10 Acid Yellow3 C.I.59040 Green No. 204 D & C Green No. 8 Solv. Green 7 C.I.60730Violet No. 401 (EXT. D & C Violet No. 2) Acid Violet No. 43 C.I.61570Green No. 201 D & C Green No. 5 Acid Green 25 C.I.73015 Blue No. 2 FD &C Blue No. 2 Acid Blue 74

Other representative examples of colorants that are usable in thecontext of the present embodiments include, natural coloring materialssuch as, but not limited to, kuchinashi blue, kuchinashi yellow,shisonin, grapeskin extract, cacao pigment, safflower yellow, hibiscuspigment, lac dye, cochineal, shikon, beet red, brazilin curcumin,riboflavin, lutein, carotenoids, annatto), paprika, carminic acid,carmin, anthocyanins, cabbage, chlorophyll, chlorophyllin,copper-chlorophyll, copper-chlorophyllin, caramel and carbomedicinalis.

The light absorbing medium can also be in the form of a film having athickness in the micrometer or sub-micrometer scale (e.g., thickness of0.5 μm) and sufficiently high light absorption fraction to elicit therequired temperature spikes in the target cells (preferably above 0.2,more preferably above 0.5, more preferably above 0.9 across a wavelengthrange spanning from IR to UV or a specific band). Representativeexamples of film materials suitable for the present embodiment include,without limitation, a carbon or graphite film, a film containingmetallic nanoparticles such as gold, silver or titanium-oxide, and afilm of Bacteriorhodopsin.

Implantation and dispersal of the light absorbing medium can be via anytechnique known in the art, including, without limitation, localinjection of a liquid buffer containing the medium to the targetlocation. For example, in embodiments in which the target location isthe retina the light absorbing medium can be injected directly next toor into the retina. In some embodiments of the present invention themedium could also be injected into the vasculature (e.g., directly orvia liposomes) for delivery of the medium to the target location viablood flow, etc. Delivery can be based on methods of targeted drugdelivery, or it may be supplemented by activation or expression of themedium, e.g., via optical activation. For example, in some embodimentsof the present invention, liposomes encapsulated with a dye areintroduced into the vasculature, for delivery of the liposome to thetarget location by the blood stream. Thereafter, the target location isilluminated by light at a wavelength selected to release the dye fromthe liposomes at the target location. When the light absorbing medium isin the form of a film, it can be implanted at the target location by aminimal invasive or a fully invasive procedure, as known in the art.

At 13 the method directs light to the artificial light absorbing medium,wherein wavelengths and intensities of the light are selected so as toheat the light absorbing medium by light absorption. In variousexemplary embodiments of the invention the heating is sufficient tostimulate neurons nearby (e.g., within about 50 μm from) the lightabsorbing medium. The light is preferably a laser light. For example,the light can be a monochromatic laser light or a combination of severalmonochromatic laser lights. Lasers which are not strictly monochromaticare also contemplated. In some embodiments of the present inventionwavelengths and intensities of the light are selected so as to inducetwo-photon absorption. This can be done, for example, using ultra-fastlight pulses (e.g., femtosecond laser pulses). The use of two-photonabsorption is particularly useful in situations in which lightscattering can prevent or reduce heating of the neurons at the targetlocation. For example, two-photon absorption can be employed when thetarget location is at the brain, and it is desired to stimulate neuronsbeneath the outer surface of the brain. When two-photon absorption isemployed the wavelength of the light can be longer than the visiblerange so as to allow better penetration of the light into buried tissueregions.

Preferably, the intensity and/or wavelength of the light is selectedsuch as to increase the temperature of the light absorbing medium by ΔT,where ΔT is from about 1° C. to about 10° C., or from about 2° C. toabout 7° C., or from about 3° C. to about 6° C., e.g., about 5° C. Invarious exemplary embodiments of the invention the intensity and/orwavelength of the light is selected such as to generate a substantiallyabrupt increase of the medium's temperature followed by a sufficientlyfast temperature drop. Such temperature change is referred to herein asa “temperature spike”.

The rise of temperature within a temperature spike is preferably over asub-millisecond to several millisecond time scale (e.g., within about 1milliseconds) but may also be up to 20 milliseconds. The drop oftemperature within a temperature spike is preferably over a fewmilliseconds time scale (e.g., a drop of about 3° C. within about 5milliseconds), but may also be much longer. A representative example ofa temperature spike is provided in FIG. 12 of the Examples section thatfollows. Under conditions of thermal confinement, a temperature spikereaches the following temperature increase:

${\Delta \; T} \approx \frac{\alpha \cdot \tau \cdot P}{\pi \; r^{2}{d \cdot c_{v}}}$

Where P (Watt) is the laser power hitting the spot during a pulse ofinterval τ (seconds), α is the fraction of incoming power absorbed bythe spot of radius r (cm) and along a depth d (cm), and c_(v)=4.2J/cm³/° C. is the specific heat capacity of water. Thermal confinementoccurs approximately when the pulse is shorter than τ<δ²/κ. where δ(cm)is the characteristic (smallest) dimension and κ=1.3×10⁻³ cm²/sec is thethermal diffusitivity of water.

The method ends at 14.

FIG. 2 is a flowchart diagram of another method for stimulating neuronspresent in a living body, according to various exemplary embodiments ofthe present invention. The method is referred to hereinunder as method20.

Method 20 begins at 21 and continues to 22 at which light is spatiallymodulated to encode a stimulation pattern therein. The light ispreferably a laser light. For example, the light can be a monochromaticlaser light or a combination of several monochromatic laser lights.Lasers which are not strictly monochromatic are also contemplated.

The stimulation pattern is in accordance with the neurological type ofthe target location at which the method is executed. For example, whenthe target location is at the retina or the visual cortex, thestimulation pattern can correspond to visual information, e.g., an imageof a scene captured by an imaging device; and when the target locationis at the cochlea or the auditory cortex, the stimulation pattern cancorrespond to acoustic information captured by an acoustical recordingdevice. Other stimulation patterns, e.g., predetermined patterns fortreating a neurological condition or disease, or for stimulating a limbto perform an action, are also contemplated.

The stimulation pattern can be two-dimensional or three-dimensional, asdesired.

The term two-dimensional stimulation pattern as used herein refers to apattern engaging a locus at the target location which is substantially asurface. Two-dimensional stimulation pattern may also penetrate, to someextend (e.g., less than 50 μm), to neural tissue beneath the surface.Two-dimensional stimulation pattern can be achieved via single-photon ortwo-photon absorption mechanisms.

The term three-dimensional stimulation pattern as used herein refers toa pattern engaging a locus at the target location which is substantiallya volume. A three-dimensional stimulation pattern penetrates at least 50μm or at least 100 μm or at least 500 μm or at least 1 mm or at least 2mm or at least 3 mm beneath the outer surface of the target location.Three-dimensional stimulation pattern can be achieved via two-photonabsorption mechanisms.

The spatial modulation of the light can be done by a spatial lightmodulator, such as, but not limited to, the spatial light modulatordisclosed in U.S. Pat. Nos. 5,073,010, 5,130,830, 5,177,628 and5,844,709, the contents of which are hereby incorporated by reference. Aspatial light modulator typically operates according to the principlesof light diffraction wherein each elementary unit (e.g., a pixel) of themodulator locally modulates the phase of a portion of a light beamimpinging thereon, to provide a predetermined light profile.

A light ray is mathematically described as a one-dimensionalmathematical object. As such, a light ray intersects any surface whichis not parallel to the light ray at a point. A light beam thereforeintersects a surface which is not parallel to the beam at a plurality ofpoints, one point for each light ray of the beam. Generally, a profileof the light beam refers to an optical characteristic (intensity, phase,frequency, brightness, hue, saturation, etc.) or a collection of opticalcharacteristics of the locus of all such intersecting points. Typically,but not obligatorily, the profile of the light beam is measured at aplanar surface which is substantially perpendicular to the propagationdirection of the light.

The locus of points at which all light rays of the beam has the samephase is referred to as the wavefront of the beam. For collimated lightbeam, for example, the wavefront is a plane perpendicular to thepropagation direction of the light, and the light is said to have aplanar wavefront.

Thus, the term “profile” is used to optically characterize the lightbeam at its intersection with a given surface, while the term“wavefront” is used to geometrically characterize a surface for a givenphase.

Since the profile, as explained, can include one or more opticalcharacteristics of a locus of points on a surface, it can be representedby one or more two-dimensional profile functions which return theoptical characteristics of a point on the surface, given thetwo-dimensional coordinates of the point. A general profile function isdenoted by Γ_(j) (ξ, η), where the index j represents the type ofoptical characteristic returned by the function (phase, intensity,frequency, etc.) and the tuple (ξ,η) represents the coordinates of apoint on the surface in an arbitrary coordinate system (Cartesian,polar, parabolic, etc.). Thus, for example, Γ_(φ)(x,y), Γ_(I)(x,y) andΓ_(v)(x,y) returns the phase φ, intensity I and frequency v of the lightat a point (x,y) in Cartesian coordinate system.

A profile relating to a specific optical characteristic is referred toherein as a specific profile and is termed using the respectivecharacteristic. Thus, the term “intensity profile” refers to theintensity of the locus of all the intersecting points, the term “phaseprofile” refers to the phase of the locus of all the intersectingpoints, the term “frequency profile” refers to the frequency of thelocus of all the intersecting points, and so on. Similarly to thegeneral profile function, a specific profile function can also berepresented by a two-dimensional function.

In various exemplary embodiments of the invention the spatial modulationis a phase-only modulation wherein only the phase varies across themodulator (i.e., non flat phase-profile), but all other opticalcharacteristics are substantially constant across the modulator. In someembodiments the spatial modulation is by means of twomodulation-subunits arranged to allow concurrent phase and amplitudemodulation of the incoming beam.

The diffraction pattern which modulates the light is also known as ahologram. And the process of forming the diffraction pattern and usingit for modulating light is often times referred to in the literature asholography. Thus, the method of the present embodiments generatesholographic data which is then reconstructed to provide the desiredstimulation pattern. It is appreciated that since the hologram is usedfor encasing the stimulation pattern, it can be a two-dimensionalobject, irrespectively of the dimensionality of the stimulation pattern.The advantage of holography is that it allows the generation ofhigh-intensity sparse stimulation patterns with relatively low opticallosses.

The method continues to 23 at which the modulated light is directed tothe target location so as to form the stimulation pattern thereat. Thelight is directed by means of optics which may include free-space optics(e.g., an arrangement of lenses, microlens arrays, diffractive elements,etc.) and/or guiding optics (e.g., waveguides, optical fibers, fiberbundles, gradient-index (GRIN) fiber lenses, lens-relay endoscopes,etc.) and/or a generalized phase contrast filter (for transforming phasemodulations into intensity modulations). When the target location is theretina of the eye, the optics may optionally include the cornea and lensof the subject. Guiding optics are particularly useful when the targetlocation is not optically accessible by direct illumination.

In some embodiments of the present invention wavelengths and intensitiesof the light are selected so as to induce two-photon absorption, asfurther detailed hereinabove.

In various exemplary embodiments of the invention the wavelengths andintensities of the modulated light are selected so as to generatesufficient heat to stimulate neurons by the stimulation pattern.Preferably, the intensity and/or wavelength of the light is selectedsuch the temperature at a spot within the stimulation pattern isincreased by ΔT, where ΔT is from about 1° C. to about 10° C., or fromabout 2° C. to about 7° C., or from about 3° C. to about 6° C., e.g.,about 5° C. In various exemplary embodiments of the invention theintensity and/or wavelength of the light is selected such as to generatea temperature spike, as further detailed hereinabove.

A temperature spike can be achieved, for example, by adjusting thewavelength and/or intensity of the modulated light so as to increaselight absorption in specific biological materials present in the targetlocation.

For example, the wavelength and/or intensity and/or spatial distributionof the modulated light can be adjusted such that the light is absorbedby Haemoglobin. Haemoglobin absorbs light from about 380 nm to about 450nm (absorption peak at about 415 nm) and from about 480 nm to about 600nm (absorption peak at about 575 nm). Haemoglobin also has a two-photonabsorption range of 820-840 nm (absorption peak at about 830 nm).

In some embodiments of the present invention the wavelength and/orintensity of the modulated light are adjusted such that the light isabsorbed by Flavoproteins which absorb light from about 400 nm to about500 nm.

In some embodiments of the present invention the wavelength and/orintensity and/or spatial distribution of the modulated light areadjusted such that the light is absorbed by Lipids (either triglycerideswhich are mainly present in subcutaneous tissues and around internalorgans, or phospholipids which are present in the cells' membrane).Lipids absorb light in the range 880 nm-970 nm.

In some embodiments of the present invention the wavelength and/orintensity of the modulated light are adjusted such that the light isabsorbed by Cytochrome c Oxidase. This is the terminal protein in theelectron chain within the inner mitochondrial membrane. It has a fewabsorption peaks in the visible and NIR range (e.g., a peak at about 850nm).

The method ends at 24.

FIG. 3 is a flowchart diagram of another method for stimulating neuronspresent in a living body, according to various exemplary embodiments ofthe present invention. The method, referred to hereinunder as method 30,combines several operations of methods 10 and 20.

Method 30 begins at 31 and, optionally and preferably, continues to 32at which a light absorbing medium is implanted in the target location,as further detailed hereinabove (see, e.g., 12 in FIG. 1 and theaccompanying description). Alternatively, the method can be executedafter the light absorbing medium has been implanted in the targetlocation. Method 30 can proceed to 33 at which the light is spatiallymodulated to encode a stimulation pattern therein, as further detailedhereinabove (see, e.g., 22 in FIG. 2 and the accompanying description).The stimulation pattern can be two-dimensional or three-dimensional, asdesired.

Method 30 can then proceed to 34 at which the modulated light isdirected to the artificial light absorbing medium, wherein wavelengthsand intensities of the modulated light are selected so as to heat thelight absorbing medium by light absorption, as further detailedhereinabove (see, e.g., 13 in FIG. 1 and the accompanying description,mutatis mutandis). In some embodiments of the present inventionwavelengths and intensities of the light are selected so as to inducetwo-photon absorption, as further detailed hereinabove.

The method ends at 35.

FIG. 4 is a flowchart diagram of another method for stimulating neuronspresent in a living body, according to various exemplary embodiments ofthe present invention. The method is referred to hereinunder as method40.

The method begins at 41 and, optionally and preferably, continues to 42at which light-activated ion-channel proteins are delivered to theneurons in the target location. Alternatively, the method can beexecuted after the light-activated ion-channel proteins are delivered tothe neurons.

Several types of light-activated ion-channel proteins are contemplated.Representative examples include, without limitation, ChR2, VChR1, NpHR,Chop2, ChR2-310, Chop2-310. In various exemplary embodiments of theinvention the light-activated ion-channel protein is ChR2(ChannelRhodopsin 2) which is a directly light-gated cation-selectiveion channel in the green algae Chlamydomonas Reinhardtii. This membranechannel opens rapidly after absorption of a blue photon, generating alarge permeability for cations, and can thus be used for depolarizingcells using illumination. Combination of several proteins is alsocontemplated. For example, in some embodiments of the present inventionChR2, VChR1(Volvox ChannelrhodopsinI) and NpHR (Halorhodopsin) aredelivered to the target location, wherein ChR2, VChR1 facilitateexcitation of neuronal activity and NpHR facilitates inhibiting neuronalactivity.

The light-activated ion-channel proteins can be delivered by a techniqueknown in the art. For example, the neurons can be transfected by genetransfer vectors (for example, viruses) capable of inducing expressionof photosensitive ion channel (for example, ChR2 ion channels). Thevectors can include a nucleic acid sequence that codes for alight-activated ion-channel protein and a cell specific promoter,wherein the targeted neurons express the protein.

For example, viral-based proteins (e.g., lentiviruses or recombinantadeno-associated viruses) can be created to target the neurons, basedupon the proteins that they uniquely express. The neurons are theninfected by the viral-based gene-transfer proteins, and begin to expressa new type of ion channel (for example ChR2), thereby becomingphotosensitive.

The method continues to 43 at which light is spatially modulated toencode a stimulation pattern therein, as further detailed hereinabove(see, e.g., 22 in FIG. 2 and the accompanying description). Thestimulation pattern can be two-dimensional or three-dimensional, asdesired.

In various exemplary embodiments of the invention the wavelengths and/orintensities of the shapes forming the stimulation pattern are selectedso as to activate the light-activated ion-channel proteins. For example,blue light spots can be used to activate ChR2 and green-yellow lightspots can be used to activate VChR1 and NpHR.

The method continues to 44 at which the modulated light is directed tothe target location so as to form the stimulation pattern thereat. Thewavelengths and intensities of the light are selected so as to activatethe light-activated ion-channel proteins. In some embodiments of thepresent invention wavelengths and intensities of the light are selectedsuch that the light-activated ion-channel proteins are activated bytwo-photon absorption. This can be done, for example, using ultra-fastlight pulses, as further detailed hereinabove.

The light is directed by means of optics which may include free-spaceoptics (e.g., an arrangement of lenses, microlens arrays, diffractiveelements, etc.) and/or guiding optics (e.g., waveguides, optical fibers,fiber bundles, gradient-index (GRIN) fiber lenses, lens-relayendoscopes, etc.) and/or a generalized phase contrast filter (fortransforming phase modulations into intensity modulations). When thetarget location is the retina of the eye, the optics may optionallyinclude the cornea and lens of the subject. Guiding optics areparticularly useful when the target location is not optically accessibleby direct illumination.

The method ends at 45.

Reference is now made to FIGS. 5A-E which are schematic illustrations ofa neurostimulation system 50, according to various exemplary embodimentsof the present invention.

System 50 can be used for stimulating neurons at a target location 52,such as, but not limited to, a retina, a cochlea, a cortex, a brainstemstructure, a spinal cord or any location in the living body in whichthere is a large population of neural tissues, as further detailedhereinabove. The target location shown in FIGS. 5A-B is a retina, thetarget location shown in FIGS. 5C-D is the cerebral cortex, and thetarget location shown in FIG. 5E is the cochlea, but it is to beunderstood that more detailed reference to a retina, cerebral cortex orcochlea is not to be interpreted as limiting the scope of the inventionin any way.

In some embodiments of the present invention, target location 52 isimplanted with an artificial light absorbing medium 54. Medium 54 can bein the form of a plurality of particles, as illustrated in FIGS. 5A, 5Band 5C or in the form of a film as illustrated in FIG. 5B. Any of theaforementioned types of light absorbing media can be implanted in targetlocation 52. In various exemplary embodiments of the invention medium 54is implanted extracellularly in target location 52. The syringe 56illustrated in FIG. 5A represents an embodiment in which medium 54 isimplanted in target location 52 by means of local injection. However,this need not necessarily be the case, since, for some applications, itmay be desired to employ other implantation techniques as furtherdetailed hereinabove. For clarity of presentation, medium 54 is notshown in FIG. 5E, but one of ordinary skill in the art would appreciatethat medium 54 may be delivered to the cochlea, if desired, by any ofthe aforementioned techniques.

System 50 comprises an illumination system 58 having a light source 60for generating light 62 and optics 64 for directing light 62 to targetlocation 52. When target location 52 is implanted with artificial lightabsorbing medium 54, wavelengths and/or intensities and/or spatialdistribution of light 62 are selected so as to heat medium 54 by lightabsorption, hence to stimulate neurons nearby medium 54, as furtherdetailed hereinabove. When target location 52 is not implanted withartificial light absorbing medium, wavelengths and/or intensities and/orspatial distribution of light 62 are selected so as to so as to increaselight absorption in specific biological materials present in the targetlocation, hence to heat the neurons and stimulate them, as furtherdetailed hereinabove. When the neurons at target location 52 aretransfected by gene transfer vectors capable of inducing expression ofphotosensitive ion channel, wavelengths and/or intensities and/orspatial distribution of light 62 are selected so as to activate thelight-activated ion-channel proteins.

Light source 60 is preferably a laser light. For example, light source60 can be a monochromatic laser light or a combination of severalmonochromatic laser lights. Lasers which are not strictly monochromaticare also contemplated. When several lasers are employed, they canoperate in synchronization (simultaneously or in a time-multiplexedmanner).

The graphs shown in FIGS. 5A-B show the temperature profile of medium 54at three different time instants, immediately following the illumination(top graph of each Figure), about 1 ms following the illumination(middle graph of each Figure) and about 5 ms following the illumination(middle graph of each Figure). As shown, in the embodiment shown in FIG.5B, the peak temperature is higher compared to the embodiment shown inFIG. 5A to insure that the temperature spike experienced by the cellswill be sufficiently high to excite them.

In various exemplary embodiments of the invention illumination system 58comprises a projector system which generates a spatially modulated lightbeam encoded with a stimulation pattern, as further detailedhereinabove. A more detailed description of a projector system isprovided hereinunder. The stimulation pattern formed at the targetlocation by the projector system can be two-dimensional orthree-dimensional. When the projector system provides a two-dimensionalstimulation pattern, it can be configured to induce a single-photon or atwo-photon absorption. When the projector system provides athree-dimensional stimulation pattern, it is preferably configured toinduce a two-photon absorption, as further detailed hereinabove.

Optics 64 can be a free-space optics and/or guiding optics, as furtherdetailed hereinabove.

With specific reference to FIG. 5C, free-space or guiding optics 64 canfocuses light 62 through a hole 68 burred in the skull directly untotarget location 52 at cerebral cortex 66. Optical stimulation of neuronsburied up to about 300 μm from the surface of the cerebral cortex 66 canbe achieved according to various exemplary embodiments of the presentinvention via linear single-photon or non-linear two-photon opticaleffects. Optical stimulation of neurons buried up to 2 mm from thesurface of the cerebral cortex, can be achieved according to variousexemplary embodiments of the present invention using ultrafast laserspulses to generate nonlinear two-photon optical effects.

With specific reference to FIG. 5D optical stimulation of neurons buriedat any distance, can be achieved according to various exemplaryembodiments of the present invention by focusing a laser outside theskull unto the entrance aperture of an optical fiber or fiber bundle 69to achieve single-point controlled excitation. Alternatively, aholographic pattern as described above can be reconstructed unto theentrance aperture of an image-preserving endoscopic device which guidesthe light through the skull and brain and then re-image the patternsonto target location 52. Devices with appropriate dimensions (mm orsub-mm) are typically prepared as bundles of fibers, gradient-index(GRIN) fibers or relay-lens boroscopes. Bundles of fibers have theadvantage of flexibility. The endoscopic device can have a lens at theimplanted end (typically a GRIN lens). The endoscopic device may also beterminated with a side-firing prism or mirror surface e.g., for reducingtissue damage.

With specific reference to FIG. 5E, when the target location is thecochlea 90, light source 60 can be placed distal to the cochlea 90 inoperation, for instance, under the scalp or within a behind-the-earhousing for a speech processor. Alternatively, the light source can bein a box positioned, for example, on the hip. Guiding optics 64 canfocus the light (not shown) to a plurality of auditory neurons 92 incochlea 90, e.g., by means of a plurality of optical fibers that areimplanted in cochlea 90 and are optically coupled to light source 60.Alternatively, side-emitting waveguides can be utilized as optics 64.

Reference is now made to FIG. 6 which is a schematic illustration aneurostimulation device 70, according to various exemplary embodimentsof the present invention. Device 70 forms a stimulation pattern attarget location 52 and can be used for stimulating neurons present atthe target location whether or not the target location is implanted withartificial light absorbing medium 54. Device 70 can also be used forstimulating neurons when the neurons express light-activated ion-channelproteins.

Device 70 comprises a projector system 72 which generates a spatiallymodulated light beam encoded with a stimulation pattern. Projectorsystem 72 can comprise one or more light sources 74 which generate readlight 82, and a spatial light modulator (SLM) 76 which performs themodulation.

Although device 70 is shown as having two light sources, this need notnecessarily be the case, since device 70 can have any number of lightsources, depending, for example, on the number of different specificwavelength bands which are required to stimulate the neurons.

Generally, SLM 76 comprises an address unit 78 and a modulation unit 80.Address unit 78 receives a pattern from an external source 88 and altersoptical characteristic of modulation unit 80. The spatial variations ofoptical characteristic across modulation unit 80 are known as ahologram. Modulation unit 80 receives and modulates read light 82 inaccordance with the hologram. Thus, SLM 76 modulates read light 82 inaccordance with the pattern signal to provide modulated light 84constituting a reconstructed stimulation pattern.

Address unit 78 can be either electrically-addressable in which case itreceives an electric pattern signal, or optically-addressable, in whichcase it receives an optical pattern signal, also known as “write light”(not shown). External source 88 can include a data processor whichcalculates the pattern and transmits it to unit 78 either as electricalsignals or as optical signals.

Modulation unit 80 can comprise a nematic liquid crystal, or aferroelectric liquid crystal (FLC), the latter being preferred from thestandpoint of high response speed. Modulation unit 80 can also comprisean array of minors or micromirrors capable of moving over a fullwavelength allowing 2π of phase control. Other MEMS based modulationunits and spatial light modulators are not excluded from the scope ofthe present invention.

In various exemplary embodiments of the invention modulation unit 80provides a modulated light beam 84 having a substantially flat intensityprofile but non-flat phase profile, as further detailed hereinabove. Invarious exemplary embodiments of the invention modulation unit 80induces a phase-only modulation, as further detailed hereinabove. Insome embodiments, the modulation unit comprises two modulation-subunitsarranged to allow concurrent phase and amplitude modulation of theincoming beam.

Spatial light modulators suitable for the present embodiments aredisclosed in U.S. Pat. Nos. 5,073,010, 5,130,830, 5,177,628 and5,844,709, the contents of which are hereby incorporated by reference.

Read light 82 from the light source(s) can be directed to modulationunit 80 of SLM 76 via one or more optical redirecting and focusingelements. In the representative example illustrated in FIG. 6, the lightbeam from a first source 74 a is redirected by a mirror M and passesthrough a dichroic mirror DM which is selected to allow transmission ofthe light from first source 74 a but reflect light from a second source74 b. Light beam from second source 74 b is therefore combined with thebeam from source 74 a at diachronic minor DM to form read light 82. Thetwo beams are expanded using a beam expander BE to impinge on apolarizing beam splitter BS which redirects the read light to modulationunit 80. The stimulation pattern is encoded in the read light bymodulation unit 80 which provides a modulated light 84. Light 84 isprojected out of projector system 72 via beam splitter BS. It is to beunderstood, that read light 82 can be directed to SLM 76 in variousother ways, all of which are known to those skilled in the art ofoptics.

The stimulation pattern formed at the target location by projectorsystem 72 can be two-dimensional or three-dimensional, as furtherdetailed hereinabove.

Device 70 can also comprise optics 86 which directs the modulated light84 to target location 52. Optics 86 can be free-space optics (e.g., anarrangement of lenses, microlens arrays, diffractive elements, etc.)and/or guiding optics (e.g., waveguides, optical fibers, fiber bundles,gradient-index (GRIN) fiber lenses, lens-relay endoscopes, etc.) and/ora generalized phase contrast filter (for transforming phase modulationsinto intensity modulations). When the target location is the retina ofthe eye, the optics may optionally include the cornea and lens of thesubject. Guiding optics are particularly useful when the target locationis not optically accessible by direct illumination. In therepresentative embodiment illustration of FIG. 6, free-space optics isshown. In this embodiment, optics 86 includes a telescope which isoptically coupled to microscope objective. The telescope and microscopeare arranged to form the reconstructed pattern on the objective L3 ofthe microscope.

Reference is now made to FIG. 7 which is a schematic illustration of aneuroprosthesis system 100, according to various exemplary embodimentsof the present invention. System 100 comprises a neurostimulation device102 and a sensing device 104. In various exemplary embodiments of theinvention the principles and operations of neurostimulation device 102are similar to the principles and operations of device 70. In someembodiments of the present invention are similar to neurostimulationdevice 102 is device 70.

Sensing device 104 serves for sensing information from the environment106 and transmitting signals 108 pertaining to the sensed information toneurostimulation device 102. Device 102 calculates a stimulation pattern(e.g., by means of a data processor as further detailed hereinabove)based on the information and directs a modulated light 84 encoding thestimulation pattern to target location 52.

Sensing device 104 can be embodied in many forms. In some embodiments ofthe present invention sensing device 104 collects visual information.For example, sensing device 104 can be an imaging device which capturesan image of a scene and transmits it to neurostimulation device 102. Inthese embodiments, the stimulation pattern corresponds to visualinformation and the target location is the retina or the visual cortex.In some embodiments of the present invention sensing device 104 collectsacoustical information. For example, sensing device 104 can include amicrophone which collects acoustic waves from the environment andconverts them to electrical signals and a transmitter which transmitsthe signals to neurostimulation device 102. In these embodiments, thestimulation pattern corresponds to the acoustic information and thetarget location is the cochlea or the auditory cortex. Other types ofsensing devices are not excluded from the scope of the presentinvention.

System 100 or part thereof can be mounted on the subject by any knowntechnique. For example, when system 100 is used to stimulate neurons inthe retina, sensing device 104 and optionally also projector system 72can be mounted on a head-up display as known in the art. Alternatively,sensing device 104 and/or projector system 72 can be miniaturized andimplanted in the eye. When system 100 is used to stimulate neurons inthe cochlea, sensing device 104 can be miniaturized and mounted in orbehind the ear and projector system 72 is miniaturized and implanted inthe cochlea.

It is expected that during the life of a patent maturing from thisapplication many relevant light modulators will be developed and thescope of the term spatial light modulator is intended to include allsuch new technologies a priori.

Attention will now be given to the advantages and potential applicationsoffered by some embodiments of the present invention.

Embodiments of the present invention can be used to drive complex neuralactivity patterns containing many action potentials (of the order ofmillions) per second.

By controlling or perturbing neuronal dynamics, the present embodimentscan be used in academic neuroscience research as well as in many medicalapplications, e.g., the treatment of a neurological condition.

Some embodiments of the present invention can be used for the treatmentof blindness. Some of the most common causes of blindness aredegenerative diseases of the outer retina, like Age-related MacularDegeneration (AMD) and Retinitis Pigmentosa (RP), globally affectingapproximately 25-30 million and 1.5 million individuals respectively.Diseases of the outer retina result in photoreceptor loss, while theinner retinal neurons and in particular the retinal ganglion cells andtheir optic nerve projections are largely maintained functional. Thepresent embodiments can be used for artificially stimulating theserelatively well-preserved nerve cells hence to provide at least partialvision restoration for the blind. In some embodiments, visionrestoration or partial vision restoration is achieved by stimulatingneurons in other locations along the visual pathway (e.g., the opticnerve, the Lateral Geniculate Nucleus or primary visual cortex).

Some embodiments of the present invention can be used for stimulatingthe auditory system of the living subject. For example, in someembodiments of the present invention a cochlear implant is provided,wherein light is used for stimulating auditory neurons to evoke actionpotentials therein. The cochlear implant can be implanted into the scalatympani of the cochlea, or in an alternate downstream location along theauditory pathway, to provide sound perception for deaf individuals.

Some embodiments of the present invention can be used for Deep-BrainStimulators (DBS) or Vagal Nerve Stimulators (VNS) for the treatment ofmany types of neurological as well as psychiatric conditions, e.g.,without limitation dystonia, epilepsy, tourette's syndrome, vegetativestate, metabolic disorder (e.g., obesity), mood disorders (e.g.,depression and bipolar disorder), anxiety disorders (e.g., generalizedanxiety disorder and obsessive-compulsive disorder), chronic pain (e.g.,visceral pain, neuropathic pain, nociceptive pain, phantom-limb pain),gastrointestinal disorders (e.g., gastroesophageal reflux disease(GERD), fecal dysfunction, gastrointestinal ulcer, gastroparesis, andother gastrointestinal motility disorders), hypertension, cardiacdisorders (e.g., tachycardia, bradycardia, other arrhythmias, congestiveheart failure, and angina pectoris), psychotic disorders (e.g.,schizophrenia), cognitive disorders, dementia (e.g., Alzheimer'sdisease, Pick's disease, and multi-infarct dementia), eating disorders(e.g., anorexia nervosa and bulimia), sleep disorders (e.g., insomnia,hypersomnia, narcolepsy, and sleep apnea), endocrine disorders (e.g.,diabetes), movement disorders (e.g., Parkinson's disease and essentialtremor), and/or headache (e.g., migraine and chronic daily headache).

DBS and VNS stimulations can be executed either for treating aparticular occurrence of a syndrome, or for treating a chroniccondition. For treatments of chronic conditions by DBS, some embodimentsof the present invention provide an implantable chronic stimulationdevice which is adapted for intra-skull implantation.

For treatments of chronic conditions by VNS, some embodiments of thepresent invention provide an implantable stimulation device which isadapted for implantation adjacent to the vagus nerve.

Some embodiments of the present invention can be used as a laboratoryequipment, e.g., for stimulating neurons in an ex-vivo neuron culture.

As used herein the term “about” refers to ±10%.

The word “exemplary” is used herein to mean “serving as an example,instance or illustration.” Any embodiment described as “exemplary” isnot necessarily to be construed as preferred or advantageous over otherembodiments and/or to exclude the incorporation of features from otherembodiments.

The word “optionally” is used herein to mean “is provided in someembodiments and not provided in other embodiments.” Any particularembodiment of the invention may include a plurality of “optional”features unless such features conflict.

The terms “comprises”, “comprising”, “includes”, “including”, “having”and their conjugates mean “including but not limited to”.

The term “consisting of means “including and limited to”.

The term “consisting essentially of” means that the composition, methodor structure may include additional ingredients, steps and/or parts, butonly if the additional ingredients, steps and/or parts do not materiallyalter the basic and novel characteristics of the claimed composition,method or structure.

As used herein, the singular form “a”, “an” and “the” include pluralreferences unless the context clearly dictates otherwise. For example,the term “a compound” or “at least one compound” may include a pluralityof compounds, including mixtures thereof.

Throughout this application, various embodiments of this invention maybe presented in a range format. It should be understood that thedescription in range format is merely for convenience and brevity andshould not be construed as an inflexible limitation on the scope of theinvention. Accordingly, the description of a range should be consideredto have specifically disclosed all the possible subranges as well asindividual numerical values within that range. For example, descriptionof a range such as from 1 to 6 should be considered to have specificallydisclosed subranges such as from 1 to 3, from 1 to 4, from 1 to 5, from2 to 4, from 2 to 6, from 3 to 6 etc., as well as individual numberswithin that range, for example, 1, 2, 3, 4, 5, and 6. This appliesregardless of the breadth of the range.

Whenever a numerical range is indicated herein, it is meant to includeany cited numeral (fractional or integral) within the indicated range.The phrases “ranging/ranges between” a first indicate number and asecond indicate number and “ranging/ranges from” a first indicate number“to” a second indicate number are used herein interchangeably and aremeant to include the first and second indicated numbers and all thefractional and integral numerals therebetween.

As used herein the term “method” refers to manners, means, techniquesand procedures for accomplishing a given task including, but not limitedto, those manners, means, techniques and procedures either known to, orreadily developed from known manners, means, techniques and proceduresby practitioners of the chemical, pharmacological, biological,biochemical and medical arts.

As used herein, the term “treating” includes abrogating, substantiallyinhibiting, slowing or reversing the progression of a condition,substantially ameliorating clinical or aesthetical symptoms of acondition or substantially preventing the appearance of clinical oraesthetical symptoms of a condition.

It is appreciated that certain features of the invention, which are, forclarity, described in the context of separate embodiments, may also beprovided in combination in a single embodiment. Conversely, variousfeatures of the invention, which are, for brevity, described in thecontext of a single embodiment, may also be provided separately or inany suitable subcombination or as suitable in any other describedembodiment of the invention. Certain features described in the contextof various embodiments are not to be considered essential features ofthose embodiments, unless the embodiment is inoperative without thoseelements.

Various embodiments and aspects of the present invention as delineatedhereinabove and as claimed in the claims section below find experimentalsupport in the following examples.

EXAMPLES

Reference is now made to the following examples, which together with theabove descriptions illustrate some embodiments of the invention in a nonlimiting fashion.

Example 1

Some embodiments of the present invention allow controlling neuronalcircuits with cellular resolution. This is useful in many medicalapplications because neighboring neurons in real circuits are oftenfound to have widely divergent response properties.

The present embodiments are advantageous over optical excitation oftraditional optogenetic populations, since traditional techniques arelimited to nonspecific population-wide flashes of light delivered usingplanar whole-field illumination or delivered to deep brain structuresusing implanted optical fibers [see, e.g., Aravanis, A. M. et al. “Anoptical neural interface: in vivo control of rodent motor cortex withintegrated fiberoptic and optogenetic technology,” J Neural Eng 4,S143-56 (2007)]. Some techniques allow patterned neural photo-excitationvia a modality known as neurotransmitter uncaging [see, e.g., Shoham,S., O'Connor, D. H., Sarkisov, D. V. & Wang, S. S.-H. “Rapidneurotransmitter uncaging in spatially defined patterns,” Nature Methods2, 837-843 (2005)]. These techniques, however, employ rapidrandom-access laser deflection, which has a very limited ability toflexibly stimulate large populations because of the relatively long,millisecond-scale, dwell time each neuron requires. More recently,parallel photo-stimulation systems using microdisplays based onmicro-mirror arrays have been developed [Reutsky, I., Ben-Shimol, D.,Farah, N., Levenberg, S. & Shoham, S. in CNE '07. 3rd InternationalIEEE/EMBS Conference on Neural Engineering, 2007 50-52 (2007); Wang, S.et al. “All optical interface for parallel, remote, and spatiotemporalcontrol of neuronal activiy,” Nano Lett 7, 3859-63 (2007)]. However,these systems require very strong light sources due to their spectacularinefficiency when used for projecting a typical sparse, intenseexcitation pattern.

The need for an optical tool which can be optimized for optogeneticphoto-stimulation of large neuron populations has led the presentinventor to devise a technique for stimulating neurons by spatial lightmodulation. The technique of the present embodiments combines parallelsimultaneous photo-stimulation of multiple locations in two or threedimensions with high intensity and efficiency that are characteristic ofsequential deflection methods.

It is noted that spatial light modulation has heretofore been employedin the field of multi-focal optical tweezers [Dufresne, E. R., Spalding,G. C., Dearing, M. T., Sheets, S. A. & Grier, D. G. Computer-generatedholographic optical tweezer arrays. Review of Scientific Instruments 72,1810-1816 (2001)].

The present Example describes experiments performed according to theteachings of the present embodiments.

The experimental setup and the procedure are schematically illustratedin FIGS. 8A-C. Polarized laser light from two sources is combined usinga dichroic minor and expanded. The expanded beam is modulated by aspatial light modulator which constitutes a hologram thereon, and imagedthrough a telescope to form a reconstruction pattern at the backaperture of a microscope objective. The reconstruction patterns in theFourier plane serve to excite or inhibit nerve cells expressinglight-gated ion channels in their cell membranes. FIG. 8B is amagnification of a portion of the nerve cells sample (see box 200 inFIG. 8A), and FIG. 8C illustrates the process of stimulation of a singleneuron (see, e.g., circle 202 in FIG. 8B). The experiments are describedin more details in the Methods section below.

In Fresnel holography, field patterns on a spatial light modulator arerelated to the resulting stimulation pattern through the Fresneltransform:

$\begin{matrix}{{E_{stim}\left( {\overset{\_}{\rho},} \right)} = {\int\limits_{}{{E_{SLM}\left( \overset{\_}{r} \right)}^{{- 2}{\pi }\frac{\overset{\_}{r} \cdot \overset{\_}{\rho}}{\lambda \; f}}^{2{\pi }\frac{{\rho}^{2}}{\lambda \; f^{2}}}{\rho}}}} \\{{= {\mathcal{F}\left\lbrack {{E_{SLM}(r)}^{2{\pi }\frac{{\rho}^{2}}{\lambda \; f_{2}}}} \right\rbrack}},}\end{matrix}$

where r=(x, y), ρ=(u, v) are the SLM and the stimulation coordinatesrespectively, z is the distance from the focal plane and ℑ is a scaledFourier transform. To generate a set of M stimulation points with the3-D coordinates {u_(m), v_(m), z_(m)} and intensity ε_(m) ², therequired field is:

$E_{SLM}^{\prime} = {\sum\limits_{m = 1}^{M}\; {ɛ_{m}^{2{\pi }\frac{{\overset{\_}{\rho}}_{m} \cdot \overset{\_}{r}}{\lambda \; f}}{^{{- 2}{\pi }\frac{z_{m}r^{2}}{\lambda \; f^{2}}}.}}}$

E′_(SLM) contains both amplitude and phase information. According tovarious exemplary embodiments of the present invention E′_(SLM) isapproximated using a phase-only modulation of an input laser beam in adiscrete pixel matrix. Phase-only modulation allows the entire incomingbeam power to be diffractively distributed between the stimulationpoints with minimal power loss. In practice, the efficiency is limitedby technical design parameters, such as the SLM reflectivity and thelike, and by the inherent approximability of arbitrary stimulationpatterns by phase-only SLM images (about 85% with continuous phasemodulation and about 30% with binary phase modulation). In the presentexample, binary phase modulation was employed.

FIG. 8D shows a phase image used to generate a typical sparsepseudo-random pattern of photo-stimulation points. For pseudo-randomstimulation patterns the SLM phase image can be computed rapidly using asingle-step Fourier transform. For inherently symmetric stimulationgrids, on the other hand, computation of the phase modulation imagetypically requires more cycles of iterative algorithms, and may havelower overall diffraction efficiency. It was found by the presentinventors that highly symmetric stimulation patterns can be achieved bypseudo-random division of stimulation points between several frames anddisplaying these frames in an alternating matter. Such procedure createsenough randomness for efficient holograms.

The spatial range of photo-stimulation loci is primarily limited by theinherent spatial sampling performed by the SLM's pixel matrix (withpitch Δ). Comparison of the required bandwidth for a single stimulationpoint (see Equation for E′_(SLM) above) to the SLM's bandwidthlimitation of 1/(2Δ) in each axis, defines a rectangular dipyramid ofaccessible locations:

${B(E)}_{x,y} = \left\{ {\begin{matrix}{{\frac{u}{\lambda \; f} + {\frac{z}{\lambda \; f^{2}}L}} \leq \frac{1}{2\Delta}} \\{{\frac{v}{\lambda \; f} + {\frac{z}{\lambda \; f^{2}}L}} \leq \frac{1}{2\Delta}}\end{matrix},} \right.$

where L is the SLM length. Outside the locus, L can be effectivelydecrease to reduce the 2^(nd) term

$\left( {L^{eff} = {\frac{\lambda \; f^{2}}{2{\Delta \cdot _{m}}} - \frac{u_{m}f}{_{m}}}} \right)$

by truncating phase patterns, at the price of bigger, less efficientspots.

FIG. 9A shows the theoretical spatial distribution of efficiency withinthe “accessible” dipyramid:

$\eta \propto {\frac{L_{x}^{eff}L_{y}^{eff}}{L^{2}}{\int\limits_{\frac{\Delta \; v}{2}}^{\frac{\Delta \; u}{2}}{\int\limits_{\frac{\Delta \; v}{2}}^{\frac{\Delta \; v}{2}}{{{F\left( E_{SLM} \right)} \cdot \sin}\; {{c\left( {v\; \Delta \; v} \right)} \cdot \sin}\; {{c\left( {u\; \Delta \; u} \right)} \cdot {u}}{v}}}}}$

where

${{\Delta \; u} = \frac{\lambda \; f}{N\; \Delta \; x}},{{\Delta \; v} = \frac{\lambda \; f}{N\; \Delta \; y}},$

and the sinc functions result from the effective filtering performed bysquare SLM pixels. This theoretical prediction is well matched withexperimental measurements of the spatial efficiency distribution alongthe x and z-axes (FIGS. 9B-C). To remove the effect of these efficiencyinhomogeneities in the x-y plane, c_(m) was set to be inverselyproportional to a spatial sinc function (FIG. 9D). The intensity of eachindividual stimulation spot can be individually controlled by varyingthe intensity ε_(m) ² in the equation for E′_(SLM). This method ofcontrol provides excellent linearity and at least 256 gray levels (FIG.9E).

The average power density at each stimulation point is inverselyproportional to the number of points simultaneously addressed. In thisembodiment, an intensity of 10 mW/mm² is approximately required toactivate the neurons. This limits the total number of simultaneouslyaccessible points to ˜250 when the guiding optics is a ×4 microscopeobjective, or more then 1000 when the guiding optics is a ×10 microscopeobjective (FIG. 9F).

A typical diameter of a nerve cell is approximately 10 μm, and theoptogenetic channels to be illuminated are distributed in a roughlyspherical geometry on the cell's outer membrane. Unlike in typicalimaging and optical tweezer scenarios, the size and Gaussian spatialdistribution of diffraction limited spots is non-optimal:

${w_{o}(z)} = {\alpha \frac{\lambda \left( {f + z} \right)}{L^{eff}}}$

In accordance with preferred embodiments of the present invention thespot size was modified by adjusting the objective effective numericalaperture, NA.

The axial resolution is determined by objective's numerical aperture.Note that there are small changes in the numerical aperture fordifferent z.

${{\Delta \; } \propto \frac{\lambda}{{NA}^{\prime^{2}}}} = \frac{\lambda}{n^{2}{\sin^{2}\left( {\arctan \left( \frac{L}{2\left( {f + } \right)} \right)} \right)}}$

The temporal resolution of the system according to various exemplaryembodiments of the present invention is determined by the SLM refreshrate. A typical figure of rise and fall time for a ferroelectric-basedSLM, is about 50 μs. The typical time-scale for neural stimulation is ofthe order of 1 millisecond. This difference in time-scale can be usedfor time-multiplexing of several lasers with different wavelengths, thusenabling the activation of different ion channels.

Since diffraction efficiency is less than unity, some of the stimulationpower is directed to unwanted areas in the stimulation volume, resultingin noise. For low-symmetry patterns, this noise is distributed fairlyuniformly.

In some embodiment of the present invention, the guiding opticsgenerates a stimulation spot of Gaussian shape with a full width at halfmaximum diameter of 5 mm with a ×10 objective (FIG. 10A). The size ofthe spot affects the system performance. The spot size can be controlledby changing various parameters of the system. For example, by modifyingthe diameter of the laser beam illuminating the SLM, the spot size canbe changed significantly (FIG. 10B, lower marks for ×10 objective, uppermarks for ×4 objective). The spot size can also be modified by axiallyshifting the entire stimulation pattern, thus modifying the objectivenumerical aperture (FIG. 10C). Large contiguous spots with possiblyarbitrary shapes can be generated by tiling individual spots adjacent toeach other. In phase-only holography, this results in speckle noise thatmay disrupt the uniformity of the spot (FIG. 10D, left image and itscorresponding section). By time averaging over shifted versions of theholograms, speckle can be eliminated and a uniform spot can be achieved(FIG. 10D, right image and its corresponding section).

The response time of our system, based on a fast ferroelectric liquidcrystal, is sub-millisecond. This enables stimulation pulses as short as0.5 ms (FIG. 10E). This response time may also be utilized for timesharing of several different wavelengths, each stimulating a differentpopulation of neurons. An example is a dual-wavelength image with 1 msecrefresh rate (FIG. 10F).

Methods Stimulation

The optical setup consisted of two DPSS lasers, a green laser (532 nm,200 mW) and a blue laser (473 nm, 50 mW) (Viasho Lasers, China). Beamexpansion was performed by a 1:4 Galilean telescope. The diameter of theexpanded beam was approximately 4 mm (FWHM). The expanded beam wasreflected by a polarizing beam splitter onto the SLM. The SLM was aferroelectric liquid-crystal micro-display (SXGA-R3, Forth DimensionDisplays, UK) with a 30° switching angle and a 13.6 μm pixel pitch.

The incoming polarized light incident on each pixel was rotated witheither a positive or negative switching angle, depending on the pixel'sstate. The polarizing beam splitter passes only the perpendicularcomponent of the outgoing light, effectively creating a phase-onlybinary hologram with [0,π] modulation. Only the central 512×512 pixelsof the SLM were used for displaying the hologram, so as to speed upcalculations, at the expense of a larger spot size.

The modulated wavefront was imaged by a Keplerian 2:1 telescope to theback aperture of a microscope objective, to avoid vignetting. Thetelescope de-magnification increased the maximal diffraction angle. Theobjective was part of an inverted microscope (TE-2000U, Nikon, Japan),and the second lens of the telescope served also as the microscope'stube-lens. The first lens of the telescope created an intermediate imageoutside the microscope, where unwanted diffraction orders were blockedby the placement of a rectangular slit. Different microscope objectivesmay be used to obtain different combinations of resolution, field andspot size. Both a ×10 (NA=0.25, 20 mm focal length) and an ×4 (NA=0.13,50 mm focal length) were used to obtain a 0.6×0.7 mm or a 1.5×1.75 mmfield, respectively.

A CCD camera (Hammamatsu, Japan) was attached to the microscope, andused to record the resulting stimulation patterns, as well asfluorescence and bright-field images of the neural specimens. This wasalso used to align the stimulation pattern with the specimen.

The binary holograms were calculated by the “Randomal Superposition”method as described in DiLeonardo et al. “Computer generation of optimalholograms for optical trap arrays,” Opt. Express 15, 1913-1922 (2007).The target stimulation pattern was multiplied by a random phase mask,and was then transformed by inverse Fourier transform. The binary phasewas obtained from the inverse transform by the following binarizationtransformation:

E _(SLM)′(x _(i) ,y _(j))=sgn[Re(E _(SLM)(x _(i) ,y _(j)))],

where x_(i),y_(j) are the coordinates of the center of the pixel.

A single 512×512 hologram was calculated in 44 ms by an Intel Core2Q9300 2.5 GHz personal computer. Numerical processing was done by MATLABsoftware (mathworks, USA).

For highly symmetric patterns, a GSW algorithm (see DiLeonardo et al.supra) was used. The hologram sequence was transferred to the SLMthrough a DVI interface. Precise control over video timing was achievedby using the Psychtoolbox extension.

Viral infection and Electrophysiology

A Sprague Dawley (SD) rat was intravitreally injected with 3 μl of anAAV2-CAG-Chop2/GFP-WPRE-BGH-polyA expression vector at a concentrationof 1.2×10¹¹ genomic particles/ml (GeneDetect Ltd).

The animal was sacrificed a month and a half post viral infection. Thetransfected retina was isolated in physiological solution and mounted ona transparent multi-electrode array (MEA, Multi Channel Systems MCSGmbh) with the fluorescent ganglion cells facing the electrodes. Theinter-electrode spacing was 200 μm with 30 μm electrode diameter for thetotal MEA area of 1.6 mm by 1.6 mm. The image was projected onto theretina from below through a ×4 microscope objective. The retina wasstimulated with vertical blue bars projected by the SLM. Each bar wasgenerated by random projection of 20 spots with intensity 0.1 mW/mm²updated every 0.5 ms. The spot size was 12 μm at FWHM. Each bar scansthe entire field of view within 1.13 seconds, moving from right to left.The stimulus was presented multiple times, once in 10 seconds.

FIG. 11A shows raw data of a single neuron response, as a function ofthe time in seconds. As shown, there are numerous response events abovethe background noise level, demonstrating the ability of the spatiallymodulated light of the present embodiments to stimulate the neuron.

FIG. 11B shows responses of three different neurons for multiple trialsof bar presentation (scale bar: 1 second). Each vertical line representsa single stimulation of the respective cell. As shown, the stimulationsof each cell are consistent, namely the neuron is repeatedly stimulatedat equivalent time instants within a trial. On the other hand, thestimulation events of different neurons occur at different time instantswithin scan cycle of the bar over the hologram. Thus, different regionsover the hologram address different neurons. This demonstrates theability of the spatially modulated light of the present embodiments togenerate patterned stimulation.

Example 2

In this Example, the thermal response of tissue was investigated bycomputer simulations. The simulations were conducted using COMSOL®simulation software (Massachusetts, USA).

The simulations were directed to the investigation of thetime-dependence of the response of small volume of tissue to heating viaoptical energy. The tissue was simulated as made of water.

FIGS. 12A-D show spatial (FIGS. 12A-C) and temporal (FIG. 12D) thermalresponse profile of a tissue of about (30 μm)³, following heating by a0.5 ms pulse of light. Spatial profiles are shown at t=0.5 ms (FIG.12A), 2 ms (FIG. 12B) and 4 ms (FIG. 12C), where t=0 corresponds topulse onset. The light was simulated as being focused onto a diameter of30 μm absorbed within a 30 μm depth in the tissue. The energydissipation per unit area was 63 mJ/cm².

As shown a temperature spike has been successfully obtained at thetarget location, demonstrating the ability of the present embodiments toinduce thermal transients in tissue. The temperature was increasedduring the light pulse reaching a maximum of about 4° C. at t=0.5 ms.The drop of temperature was exponential with a drop of about 3° C.within the first 5 milliseconds.

Example 3

In this Example, the ability to inject a light absorbing medium next toor into the retina in vivo was demonstrated.

An Albino SD rat (300 gr) was anesthetized and 5 μl of India ink wereinjected intravitrealy into the eyeball in-vivo. The animal wassacrificed 20 min later and the eyeball was enucleated and placed in 4%paraformaldehyde for fixation and slicing.

FIG. 13 is an image of a 20 μm thick slice of the retina. Shown in FIG.13 are the photoreceptors, the inner retina with the retinal ganglioncells and the India ink stain.

Although the invention has been described in conjunction with specificembodiments thereof, it is evident that many alternatives, modificationsand variations will be apparent to those skilled in the art.Accordingly, it is intended to embrace all such alternatives,modifications and variations that fall within the spirit and broad scopeof the appended claims.

All publications, patents and patent applications mentioned in thisspecification are herein incorporated in their entirety by referenceinto the specification, to the same extent as if each individualpublication, patent or patent application was specifically andindividually indicated to be incorporated herein by reference. Inaddition, citation or identification of any reference in thisapplication shall not be construed as an admission that such referenceis available as prior art to the present invention. To the extent thatsection headings are used, they should not be construed as necessarilylimiting.

What is claimed is:
 1. A method of stimulating neurons present in aliving body, the method comprising: capturing an image of a scene;generating a hologram corresponding to a neuron stimulation patternbased on said image; and projecting said hologram onto a target locationhaving neurons therein so as to selectively stimulate said neurons. 2.The method of claim 1, wherein said target location is implanted with anartificial light absorbing medium, and wherein wavelengths andintensities of said hologram are selected so as to heat said lightabsorbing medium by light absorption.
 3. The method of claim 2, furthercomprising extracellularly implanting said artificial light absorbingmedium at said target location.
 4. The method of claim 2, wherein saidlight absorbing medium is distributed extracellularly at said targetlocation.
 5. The method of claim 1, wherein said neurons express alight-activated ion channel protein, and wherein wavelengths andintensities of said holograms are selected so as to activate saidlight-activated ion channel protein.
 6. The method of claim 5, furthercomprising transfecting said neurons by gene transfer vectors capable ofinducing expression of said light-activated ion channel protein.
 7. Themethod of claim 1, wherein said stimulation pattern is athree-dimensional stimulation pattern.
 8. The method of claim 1, whereinsaid target location is a retina.
 9. The method of claim 1, wherein saidtarget location is a cochlea.
 10. The method of claim 1, wherein saidtarget location is in the cerebral cortex.
 11. The method of claim 1,wherein said target location is in the brainstem.
 12. The method ofclaim 1, wherein said target location is the vagus nerve.
 13. The methodof claim 1, wherein said target location is a cranial nerve.
 14. Themethod of claim 1, wherein said target location is a neuron culture. 15.A neurostimulation system, comprising: an imaging device configured forcapturing an image of a scene; an illumination system, configured forgenerating a hologram corresponding to a neuron stimulation patternbased on said image; and optics configured for projecting said hologramonto a target location having neurons therein so as to selectivelystimulate said neurons.
 16. The system of claim 15, wherein saidillumination system is configured for providing phase-only modulation.17. The system of claim 15, wherein said illumination system isconfigured for providing concurrent phase and amplitude modulation. 18.The system of claim 15, wherein said optics is a free-space optics. 19.The system of claim 15, wherein said optics comprises an optical fiberbundle.
 20. The system of claim 15, wherein said stimulation pattern isa three-dimensional stimulation pattern.